US4209587A - Production of tumor angiogenesis factor by cell culture - Google Patents

Production of tumor angiogenesis factor by cell culture Download PDF

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US4209587A
US4209587A US05/974,408 US97440878A US4209587A US 4209587 A US4209587 A US 4209587A US 97440878 A US97440878 A US 97440878A US 4209587 A US4209587 A US 4209587A
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taf
cells
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William R. Tolbert
Joseph Feder
Mau-Jung Kuo
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Monsanto Co
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • C07K14/515Angiogenesic factors; Angiogenin
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/948Microorganisms using viruses or cell lines

Definitions

  • This invention relates to the in vitro production of human tumor angiogenesis factor from the human liver adenocarcinoma cell line SK-HEP-1.
  • TAF tumor angiogenesis factor
  • TAF finds further use in the development of tests such as an angiogenenic assay or a diagnostic screening test for neoplasia, Klagsbrun et al., Cancer Res. 36, 110-14 (1976); and Brem et al., Science 195, 880-81 (1977); Cancer 41, 239-44 (1978).
  • TAF also has been proposed as useful for wound healing. Rettusa et al, FASEB, Abstract No. 4309, 61st Ann. Meet., Apr. 1-8, 1977, Chicago, Ill.
  • WI-38 embryonic lung, SV W126 (SV 40 virus transformed W126), glioblastoma, meningioma and HeLa cells (the latter only in suspension culture and not in monolayers) grown in T-75 tissue culture flasks were described as giving a positive TAF response as measured by bioassay on the chick chorioallantoic membrane (CAM).
  • CAM chick chorioallantoic membrane
  • tumor cells are derived from fresh tumors or primary cultures except the WI-38, SV W126 and HeLa cells.
  • Fresh tumors and primary cultures are not, however, generally suitable sources of TAF except for limited research purposes or small scale production.
  • production from a suitable established cell line is deemed necessary.
  • the cell line should have not only specific TAF activity, but it should also have good cell growth characteristics in terms of rapid growth, good suspension growth, adaptability to large-scale culture and economical nutrient requirements.
  • cell line and "established cell line” are used herein in conformance with the definitions published by Federoff in the Tissue Culture Association Manual, Vol. 1. No. 1, pp. 53-57 (1975).
  • SK-HEP-1 human liver adenocarcinoma cell line SK-HEP-1.
  • the established cell line SK-HEP-1 was derived from a liver adenocarcinoma by G. Trempe of Sloan-Kettering Institute for Cancer Research in 1971, and cultures of the cell line are available from that institute.
  • This cell line has been known to produce tumors in nude mice but it has not heretofore been known as a suitable source of TAF.
  • Prior publications on the establishment and characterization of cell line SK-HEP-1 include the following:
  • the medium used by applicants for maintenance and growth of this cell line was Dulbecco's modification of Eagle's minimum essential medium (MEM) containing 4 mg/ml of glucose.
  • MEM Eagle's minimum essential medium
  • the culture medium was supplemented with 15% by volume fetal bovine serum without addition of any antibiotics, although lower concentrations of fetal bovine serum, for example, about 10-15%, also can be used.
  • the cells were first grown at 37° C. in attached culture in 75 cm 2 T-flasks (Falcon plastics) and were then converted to agitated, liquid suspension culture in the same medium in 4-liter and 100-liter vessels in accordance with the procedure described in U.S. Pat. No. 4,059,485. The cells were then harvested after times ranging from 5 to 11 days growth since inoculation, and TAF was extracted and assayed according to the general procedures described in U.S. Pat. No. 4,059,486.
  • the cells after growth were washed successively three times in lactated Ringer's solution, resuspended in phosphate buffered saline (pH 7-7.4) and stirred for 4 hours at 4° C.
  • the supernatant after removal of the cells by centrifugation was retained as the cell extract.
  • the cells pellet from the extract was resuspended in distilled water and stirred at 37° C. for 20 minutes.
  • the supernatant after removal of the cell debris by centrifugation was retained as the cell lysate.
  • CM-Sephadex® Pulcoa ion exchange chromatography
  • lyophilized The purified material also gave a positive test in the CAM bioassay for TAF.
  • CM-Sephadex chromatography for obtaining a TAF active fraction from tumor cells is described by Tuan et al., Biochemistry 12 (17), 3159-65 (1973).
  • nutrient culture media for culture of the SK-HEP-1 cells can be used in place of Dulbecco's MEM, for example, any of the well-known tissue culture media described by H. J. Morton, In Vitro 6, 89-108 (1970).
  • These conventional culture media contain known essential amino acids, mineral salts, vitamins and carbohydrates. They are also frequently fortified with mammalian sera such as fetal bovine serum. Suitable growth of the cells can be carried out at about 35°-38° C. but cell proliferation is best at 37° C. Growth under these cell culture conditions for about 4-8 days generally is sufficient to produce the desired TAF.
  • TAF-containing fractions can be separated and purify from the cells and cell culture product other than the illustrative specific recovery procedures described above.
  • These various recovery means include the known techniques for the separation and purification of proteinaceous substances in general such as, for example, dialysis, salt and solvent precipitation, adsorption with gels, cellulose ion exchange chromatography, Sephadex gel filtration, electrophoresis, and lyophilization.
  • TAF can be obtained by extraction from the cells followed by subjecting the extract to dialysis against NaCl (0.15M), Sephadex G-100 chromatography, dialysis against water and lyophilization analagous to the methods described by Folkman et al., J. Exptl.
  • TAF is a proteinaceous substance having a molecular weight of approximately 100,000. It will be appreciated, however, that the aforesaid molecular weights are estimates based on partially purified TAF-containing products and the inventors are not bound by such estimates or by any particular purification procedure or TAF purity.

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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Process for the production of human TAF in vitro comprising growing the human liver adenocardinoma cell line SK-HEP-1 in agitated, liquid suspension of nutrient culture medium at about 35°-38° C. for a sufficient time to elaborate TAF and recovering the resulting TAF from the cells or cell product.

Description

BACKGROUND OF THE INVENTION
This invention relates to the in vitro production of human tumor angiogenesis factor from the human liver adenocarcinoma cell line SK-HEP-1.
It has been known for some time that unless a solid tumor is provided with blood vessels by its host it remains small and dormant. The substance that is released by tumors and provides vascularization has been named tumor angiogenesis factor (TAF) by Dr. Judah Folkman of the Harvard Medical School. J. Exptl. Med. 133, 275-88 (1971); Cancer Res. 34, 2109-13 (1974).
Ever since it was recognized that tumor induced neovascularization represents the breaching of an important barrier in the control of tumor growth, considerable effort has been spent in searching for ways to inhibit neovascularization. It was early suggested by Dr. Folkman that blockade of this factor (inhibition of angiogenesis) might arrest solid tumors at a tiny diameter of a few millimeters (avascular phase). New Engl. J. Med. 285, 1182 (1971); J. Exptl. Med. 133, 275-88 (1971). One suggested approach was the raising of antibodies against TAF extracts for the production of an anti-serum. Folkman, Ann Surg. 175, 409-16 (1972); Phillips et al., Int. J. Cancer, 17, 549-58 (1976). Another approach to inhibiting neovascularization which has received much attention recently consists in treatment of tumor cells with extracts of cartilage. Eisenstein et al, Am J. Pathol. 73, 765-74 (1973); Sorgente et al., Lab. Invest. 32, 217-22 (1975); Eisenstain et al., Amer. J. Pathol. 81, 337-48 (1975; "Medical News", JAMA 232 (1), 14-15 (1975); Brem and Folkman, J. Exptl. Med. 141, 427-39 (1975); and Kuettner et al., U.S. Pat. No. 4,042,457. These efforts are limited, of course, to the amount of TAF which is available and suitable for test purposes.
TAF finds further use in the development of tests such as an angiogenenic assay or a diagnostic screening test for neoplasia, Klagsbrun et al., Cancer Res. 36, 110-14 (1976); and Brem et al., Science 195, 880-81 (1977); Cancer 41, 239-44 (1978).
TAF also has been proposed as useful for wound healing. Rettusa et al, FASEB, Abstract No. 4309, 61st Ann. Meet., Apr. 1-8, 1977, Chicago, Ill.
Various human tumor cells have been reported heretofore as capable of elaborating TAF when measured by certain assays. Thus, extracts from human neuroblastoma, Wilms' tumor and human hepatoblastoma were found to contain TAF which was able to cause the formation of new blood vessels in the subcutaneous fascia of rats. Folkman et al., J. Exptl. Med. 133, 275-88 (1971).
So also, WI-38 embryonic lung, SV W126 (SV 40 virus transformed W126), glioblastoma, meningioma and HeLa cells (the latter only in suspension culture and not in monolayers) grown in T-75 tissue culture flasks were described as giving a positive TAF response as measured by bioassay on the chick chorioallantoic membrane (CAM). Folkman and Klagsbrun, Chapter 31 of "Fundamental Aspects of Neoplasia", at page 402, (Gottlieb et al, eds.), Springer-Verlag, New York, 1975.
Subsequently, extracts from hypernephroma, haemangioma and human kidney were similarly described as eliciting a TAF response which resulted in the growth of new capillaries in the subcutaneous fascia of rats. Phillips et al., Int. J. Cancer, 17, 549-58 (1976).
In still other experiments, Hubler and Wolf demonstrated TAF responses from human cutaneous melanoma implanted in transparent hamster-cheekpouch chambers. Cancer 38, 187-92 (1976).
All of the foregoing tumor cells are derived from fresh tumors or primary cultures except the WI-38, SV W126 and HeLa cells. Fresh tumors and primary cultures are not, however, generally suitable sources of TAF except for limited research purposes or small scale production. In order to provide a commercially significant source of TAF in terms of ready availability and adequate supply, production from a suitable established cell line is deemed necessary. As a practical matter, the cell line should have not only specific TAF activity, but it should also have good cell growth characteristics in terms of rapid growth, good suspension growth, adaptability to large-scale culture and economical nutrient requirements.
The terms "cell line" and "established cell line" are used herein in conformance with the definitions published by Federoff in the Tissue Culture Association Manual, Vol. 1. No. 1, pp. 53-57 (1975).
DESCRIPTION OF THE INVENTION
Applicants have investigated numerous human tumor cell lines for the production of TAF, but most of them have been eliminated as unsuitable candidates in view of their poor growth characteristics as above-defined.
One cell line that has now been found to have good growth characteristics in suspension culture and is able to elaborate the desired TAF in suitable quantities is the human liver adenocarcinoma cell line SK-HEP-1. The established cell line SK-HEP-1 was derived from a liver adenocarcinoma by G. Trempe of Sloan-Kettering Institute for Cancer Research in 1971, and cultures of the cell line are available from that institute. This cell line has been known to produce tumors in nude mice but it has not heretofore been known as a suitable source of TAF. Prior publications on the establishment and characterization of cell line SK-HEP-1 include the following:
Fogh and Trempe, "New Human Tumor Cell Lines" in Human Tumor Cells In Vitro (Fogh. ed.), Plenum Publ. Corp., New York, 1975, pp. 115-54;
Fogh et al., J. Natl. Cancer Inst. 58, 209-14 (1977; and
Fogh et al., J. Natl. Cancer Inst. 58, 209-14 (1977); Fogh et al., N. Natl. Cancer Inst. 59, 221-25 (1977).
Initially, the medium used by applicants for maintenance and growth of this cell line was Dulbecco's modification of Eagle's minimum essential medium (MEM) containing 4 mg/ml of glucose. The culture medium was supplemented with 15% by volume fetal bovine serum without addition of any antibiotics, although lower concentrations of fetal bovine serum, for example, about 10-15%, also can be used. The cells were first grown at 37° C. in attached culture in 75 cm2 T-flasks (Falcon plastics) and were then converted to agitated, liquid suspension culture in the same medium in 4-liter and 100-liter vessels in accordance with the procedure described in U.S. Pat. No. 4,059,485. The cells were then harvested after times ranging from 5 to 11 days growth since inoculation, and TAF was extracted and assayed according to the general procedures described in U.S. Pat. No. 4,059,486.
Specifically, the cells after growth were washed successively three times in lactated Ringer's solution, resuspended in phosphate buffered saline (pH 7-7.4) and stirred for 4 hours at 4° C. The supernatant after removal of the cells by centrifugation was retained as the cell extract. The cells pellet from the extract was resuspended in distilled water and stirred at 37° C. for 20 minutes. The supernatant after removal of the cell debris by centrifugation was retained as the cell lysate.
In various runs of 4 liters and 100 liters the cell extracts and cell lysates were assayed to contain the following amounts of total protein (Lowry protein assay):
______________________________________                                    
        Suspension   Time Since                                           
        Volume       Inoculation Total                                    
Run No. (Liters)     (Days)      Protein                                  
______________________________________                                    
1       4            7           *extract                                 
                                 196 mg lysate                            
2       4            7           10.4 mg extract                          
                                 168 mg lysate                            
3       4            6           13.7 mg extract                          
                                 170 mg lysate                            
4       4            11          12.7 mg extract                          
                                 246 mg lysate                            
5       100          6           0.49 gm extract                          
                                 3.39 gm lysate                           
6       100          5           0.56 gm extract                          
                                 5.7 gm lysate                            
______________________________________                                    
 *Extract not assayed this run for total protein                          
The cell extracts from the 4-liter runs gave strongly positive tests in the bioassay for TAF in the chorioallantoic membrane (CAM) of chick embryos, in accordance with the procedure described by Folkman, Cancer Res. 34, 2109-13; 36, 110-14 (1976).
The cell extracts from the 100-liter runs were subjected to further purification by CM-Sephadex® (Pharmacia) ion exchange chromatography and lyophilized. The purified material also gave a positive test in the CAM bioassay for TAF. The use of CM-Sephadex chromatography for obtaining a TAF active fraction from tumor cells is described by Tuan et al., Biochemistry 12 (17), 3159-65 (1973).
It will be appreciated that other nutrient culture media for culture of the SK-HEP-1 cells can be used in place of Dulbecco's MEM, for example, any of the well-known tissue culture media described by H. J. Morton, In Vitro 6, 89-108 (1970). These conventional culture media contain known essential amino acids, mineral salts, vitamins and carbohydrates. They are also frequently fortified with mammalian sera such as fetal bovine serum. Suitable growth of the cells can be carried out at about 35°-38° C. but cell proliferation is best at 37° C. Growth under these cell culture conditions for about 4-8 days generally is sufficient to produce the desired TAF.
Other suitable equipment and procedures for growing cells in agitated, liquid suspension which can be adapted for culture of the cell line SK-HEP-1 will be readily apparent to the person skilled in the art by reference to well-known texts on cell culture such as, for example, Paul, "Cell and Tissue Culture," The Williams and Wilkins Company, Baltimore, 4th ed. 1970, at pages 277-291; Kruse and Patterson, "Tissue Culture Methods and Applications, " Academic Press, New York, 1973, at pages 333-377.
It should also be understood that various means can be used to separate and purify TAF-containing fractions from the cells and cell culture product other than the illustrative specific recovery procedures described above. These various recovery means include the known techniques for the separation and purification of proteinaceous substances in general such as, for example, dialysis, salt and solvent precipitation, adsorption with gels, cellulose ion exchange chromatography, Sephadex gel filtration, electrophoresis, and lyophilization. Thus, TAF can be obtained by extraction from the cells followed by subjecting the extract to dialysis against NaCl (0.15M), Sephadex G-100 chromatography, dialysis against water and lyophilization analagous to the methods described by Folkman et al., J. Exptl. Med. 133, 275-88 (1971) and Phillips et al., Int. J. Cancer 17, 549-58 (1976). According to Phillips et al, id, when the dialysate is subjected to chromatography on a column of Sephadex G-100 in 0.15 M NaCl, the fractions between 35,000 and 300,000 molecular weight contain the greatest TAF activity. According to Folkman, Cancer Res. 34, 2109-13 (1974), TAF is a proteinaceous substance having a molecular weight of approximately 100,000. It will be appreciated, however, that the aforesaid molecular weights are estimates based on partially purified TAF-containing products and the inventors are not bound by such estimates or by any particular purification procedure or TAF purity.
Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. All such further examples are included within the scope of the appended claims.

Claims (6)

What is claimed is:
1. Process for the production of human TAF in vitro comprising growing the human liver adenocarcinoma cell line SK-HEP-1 in agitated, liquid suspension of nutrient culture medium at about 35°-38° C. for a sufficient time to elaborate TAF and recovering the resulting TAF from the cells or cell product.
2. The process of claim 1 in which the nutrient culture medium in Dulbecco's modification of Eagle's minimum essential medium.
3. The process of claim 1 in which the nutrient culture medium is fortified with mammalian serum.
4. The process of claim 1 in which the nutrient culture medium is fortified with from about 10% to about 15% fetal bovine serum.
5. The process of claim 1 in which the TAF is recovered by extraction from the cells.
6. The process of claim 1 in which the nutrient culture medium is Dulbecco's modification of Eagle's minimum essential medium and is fortified with fetal bovine serum and in which the TAF is recovered by extraction from the cells.
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4296201A (en) * 1978-08-19 1981-10-20 Behringwerke Aktiengesellschaft Methods for the detection of antimitochondrial and antinuclear antibodies
WO1981003663A1 (en) * 1980-06-12 1981-12-24 Wistar Inst Human hepatoma derived cell line,process for preparation thereof,and uses therefor
EP0048202A2 (en) * 1980-09-11 1982-03-24 Merck & Co. Inc. Method of preparing a mycoplasma-free hepatoma cell line
EP0058857A2 (en) * 1981-02-24 1982-09-01 Gérard E. Renoux Hepatosin, process for preparing it and therapeutical composition containing hepatosin
EP0220241A1 (en) * 1985-04-17 1987-05-06 Harvard College Purified protein having angiogenic activity and methods of preparation.
EP0226181A2 (en) * 1985-12-17 1987-06-24 Synergen, Inc. Human placenta angiogenic factor capable of stimulating capillary endothelial cell protease synthesis, DNA synthesis and migration
US4699788A (en) * 1984-08-20 1987-10-13 Trustees Of Boston University Angiogenic factor methods of extraction and method for producing angiogenesis
US4752578A (en) * 1986-04-11 1988-06-21 Monsanto Company Collagenase inducing factor
US4994559A (en) * 1985-12-17 1991-02-19 Synergen, Inc. Human basic fibroblast growth factor
US5026839A (en) * 1985-12-17 1991-06-25 Synergen, Inc DNA encoding a basic fibroblast growth factor

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4059486A (en) * 1976-11-03 1977-11-22 Monsanto Company Cell culture process
US4059485A (en) * 1976-11-03 1977-11-22 Monsanto Company Adaptation of cell lines to suspension culture

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4059486A (en) * 1976-11-03 1977-11-22 Monsanto Company Cell culture process
US4059485A (en) * 1976-11-03 1977-11-22 Monsanto Company Adaptation of cell lines to suspension culture

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Fogh et al., J. Natl. Cancer Inst. 59, 221-225, (1977). *
Folkman et al., Fundamental Aspects of Neoplasia, pp. 401-412, Springer-Verlag, New York, (1975). *
Folkman et al., J. Exptl. Med., 133, 275-288, (1971). *
Hubler et al., Cancer 38, 187-192, (1976). *
Phillips et al., Int. J. Cancer 17, 549-558, (1976). *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4296201A (en) * 1978-08-19 1981-10-20 Behringwerke Aktiengesellschaft Methods for the detection of antimitochondrial and antinuclear antibodies
US4393133A (en) * 1980-06-12 1983-07-12 The Wistar Institute Of Anatomy And Biology Human hepatoma derived cell line, process for preparation thereof, and uses therefor
WO1981003663A1 (en) * 1980-06-12 1981-12-24 Wistar Inst Human hepatoma derived cell line,process for preparation thereof,and uses therefor
EP0048202A2 (en) * 1980-09-11 1982-03-24 Merck & Co. Inc. Method of preparing a mycoplasma-free hepatoma cell line
EP0048202A3 (en) * 1980-09-11 1983-06-01 Merck & Co. Inc. Method of preparing a mycoplasma-free hepatoma cell line
EP0058857A3 (en) * 1981-02-24 1983-11-09 Newport Pharmaceuticals International, Inc. Hepatosin, process for preparing it and therapeutical composition containing hepatosin
EP0058857A2 (en) * 1981-02-24 1982-09-01 Gérard E. Renoux Hepatosin, process for preparing it and therapeutical composition containing hepatosin
US4699788A (en) * 1984-08-20 1987-10-13 Trustees Of Boston University Angiogenic factor methods of extraction and method for producing angiogenesis
EP0220241A1 (en) * 1985-04-17 1987-05-06 Harvard College Purified protein having angiogenic activity and methods of preparation.
EP0220241A4 (en) * 1985-04-17 1988-09-28 Harvard College Purified protein having angiogenic activity and methods of preparation.
EP0226181A2 (en) * 1985-12-17 1987-06-24 Synergen, Inc. Human placenta angiogenic factor capable of stimulating capillary endothelial cell protease synthesis, DNA synthesis and migration
EP0226181A3 (en) * 1985-12-17 1989-03-08 Synergen, Inc. Human placenta angiogenic factor capable of stimulating capillary endothelial cell protease synthesis, dna synthesis and migration
US4994559A (en) * 1985-12-17 1991-02-19 Synergen, Inc. Human basic fibroblast growth factor
US5026839A (en) * 1985-12-17 1991-06-25 Synergen, Inc DNA encoding a basic fibroblast growth factor
US4752578A (en) * 1986-04-11 1988-06-21 Monsanto Company Collagenase inducing factor

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